1. Field of the Invention
The field of this invention is proteins which regulate cell function, and in particular, antagonize bone morphogenic proteins.
2. Background
Natural regulators of cellular growth, differentiation and function have provided important pharmaceuticals, clinical and laboratory tools, and targets for therapeutic intervention. A variety of such regulators have been shown to have profound effects on basic cellular differentiation and developmental pathways. For example, the recently cloned cerberus protein induces the formation of head structures in anterior endoderm of vertebrate embryos. Similarly, the noggin protein induces head structures in vertebrate embryos, and can redirect mesodermal fates from ventral fates, such as blood and mesenchyme, to dorsal fates such as muscle and notochord and can redirect epidermal fates to anterior neural fates. The activities of chordin are similar to those of noggin, reflecting a common mechanism of action; namely antagonizing bone morphogenic proteins (BMP) and thereby preventing their function. BMPs have diverse biological activities in different biological contexts, including the induction of cartilage and bone, connective tissue, and roles in kidney, tooth, gut skin and hair development.
Different members of the TGFxcex2 superfamily can instruct cells to follow different fates, for example TGFxcex2 induces neural crest to form smooth muscle, while BMP2 induces the same cells to become neurons. In Xenopus experiments, dissociated animal cap cells (prospective ectoderm) become epidermis in response to BMP4 but become mesoderm in response to activin. Since the identity between activin and BMP4 is low, it is not surprising that they induce different fates. It is more surprising that members of the BMP subfamily, which are quite closely related in sequence, can induce distinct fates. A striking example results from implantation of a matrix impregnated with a BMP into muscle; when the effects are monitored histologically, BMP2, 4 and 7 induce endochondral bone formation, whereas a related molecule BMP 12/GDF7 induces connective tissue similar to tendon. Similarly, BMP4 can induce cell death in the hindbrain neural crest, while the related protein dorsalin does not.
Since different BMP family members can induce different fates, then BMP antagonists that have specificity in blocking subsets of BMPs could change the balance of BMPs that are presented to a cell, thus altering cell fate. In view of the importance of relative BMP expression in human health and disease, regulators of cellular function and BMP function in particular; such as noggin and cerberus, provide valuable reagents with a host of clinical and biotechndlogical applications.
The present invention relates to a new family of regulators of cellular function; in particular, we have identified a new dorsalizing factor Gremlin. Gremlin""s activities are similar to those of Noggin and Chordin, however, it has no sequence similarity to them, and it is not expressed during gastrulation. Instead, zygotic Gremlin expression begins in the migrating neural crest, highlighting a potential role for organizer-like activities in the development of this cell lineage. Furthermore, we have identified a structurally and functionally-related family of proteins that includes Gremlin, the head-inducing factor Cerberus and the tumor suppressor DAN (Ozaki and Sakiyama, 1993; Bouwmeester et al., 1996). We have named this family the DAN family for the first member identified. Cerberus is expressed in the anterior endomesoderm of the gastrula organizer and has been proposed to participate in head induction (Bouwmeester et al., 1996). DAN was isolated as a putative zinc-finger protein 4downregulated in transformed cells and was subsequently shown to have tumor suppressor activity (Ozaki and Sakiyama, 1993; Ozaki and Sakiyama, 1994). Similarly, the rat homolog of Gremlin, drm, has also been proposed to have a role in controlling cell growth and differentiation (Topol et al., 1997). We demonstrate that all members of the DAN family act as BMP antagonists, while Cerberus alone blocks the activity of Activin and Nodal-like activities. We also show that DAN-family members are secreted proteins that bind BMP-2. Together, our data reveal that individual DAN-family members block the activity of specific TGF-xcex2 ligands by binding them and preventing them from interacting with their cell surface receptors.
Altschul, S. F., et al. (1990) J Mol Biol 215, 403-10.
Badley, J. E., et al. (1988) Biotechniques 6, 114-6.
Baker, J. C., and Harland, R. M. (1996) Genes and Development 10, 1880-1889.
Belo, J. A., et al. (1997) Mechanisms Of Development 68, 45-57.
Bolce, M. E., et al. (1992) Development 115, 681-8.
Bouwmeester, T., et al. (1996) Nature 382, 595-601.
Clement, J. H., et al. (1995)Mech Dev 52, 357-70.
Collazo, A., Bronner-Fraser, M., and Fraser, S. E. (1993) Development 118, 363-76.
Dale, L., et al. (1992) Development 115, 573-85.
Estevez, M., et al. (1993) Nature 365, 644-9.
Fainsod, A et al. (1994) EMBO J. 13, 5015-5025.
Gimlich, R. L., and Gerhart, J. C. (1984) Developmental Biology 104, 117-130.
Harland, R. M., and Gerhart, J. C. (1997) In Ann reviews Cell and Dev Biol, pp. 611-667.
Hemmati-Brivanlou, A., et al. (1994) Cell 77, 283-295.
Hemmati-Brivanlou, A., and Thomsen, G. H. (1995)-4. Dev Genet 17, 78-89.
Hogan, B. L., et al. (1994) Dev Suppl, 53-60.
Holley, S. A., et al. (1996) Cell 86, 607-17.
Jones, C. M., et al. (1995) Development 121, 3651-62.
Jones, C. M., et al. (1992) Development 115, 639-47.
Joseph, E. M., and Melton, D. A. (1997) Dev Biol 184, 367-72.
Kao, K. R., and Elinson, R. P. (1988) Devi Biol. 127, 64-77.
Kintner, C. R., and Brockes, J. P. (1984) Nature 308, 67-69.
Kintner, C. R., and Melton, D. A. (1987) Development 99, 311-25.
Krotoski, D. M., et al. (1988) Dev Biol 127, 119-32.
Lamb, T. M., et al. (1993) Science 262, 713-8.
Lemaire, P., Garrett, N., and Gurdon, J. B. (1995) Cell 81, 85-94.
Lennon, G., et al. (1996) Genomics 33, 151-2.
Ozaki, T., and Sakiyama, S. (1993) Proc Natl Acad Sci U S A 90, 2593-7.
Ozaki, T., and Sakiyama, S. (1994) Cancer Res 54, 646-8.
Peng, H. B. (1991) In Methods in Cell Biology 36, B.K. Kay, H.B. Peng, eds., pp. 661-662.
Piccolo, S., Sasai, Y., Lu, B., and De Robertis, E. M. (1996) Cell 86, 589-98.
Ren, P., et al. (1996) Science 274, 1389-91.
Sadaghiani, B., and Thiebaud, C. H. (1987) Dev Biol 124, 91-110.
Sasai, Y., and De Robertis, E. M. (1997) Dev Biol 182, 5-20.
Sasai, Y., et al. (1995) Nature 376, 333-336.
Sasai, Y., et al. (1994) Cell 79, 779-790.
Savage, C., et al. (1996) Proc Natl Acad Sci U S A 93, 790-4.
Schmidt, J. E., et al. (1995) Developmental Biology 169, 37-50.
Smith, J. C., et al. (1990) Nature 345, 729-31.
Smith, J. C., et al. (1991) Cell 67, 79-87.
Smith, W. C., and Harland, R. M. (1992) Cell 70, 829-40.
Smith, W. C., and Harland, R. M. (1991) Cell 67, 753-765.
Smith, W. C., Knecht, A. K., Wu, M., and Harland, R. M. (1993) Nature 361, 547-9.
Smith, W. C., McKendry, R., Ribisi, S., and Harland, R. M. (1995) Cell 82, 37-46.
Thies, R. S., et al. (1992) Endocrinology 130, 1318-24.
Thomas, P., et al. (1997) Cold Spring Harbor Symposia on Quantitative Biology 62.
Thomsen, G., et al. (1990) Cell 63, 485-93.
Thomsen, G. H., and Melton, D. A. (1993) Cell 74, 433-441.
Topol, L. Z., et al. (1997) Molecular and Cellular Biology 17, 4801-4810.
Turner, D. L., and Weintraub, H. (1994) Genes Devel. 8, 1434-1447.
Varlet, I., Collignon, J., and Robertson, E. J. (1997) Development 124, 1033-44.
Vize, P. D., Jones, E. A., and Pfister, R. (1995) Dev Biol 171, 531-40.
Wieser, R., Wrana, J. L., and Massague, J. (1995) Embo J 14, 2199-208.
Wilson, P. A., and Hemmati-Brivanlou, A. (1995) Nature 376, 331-3.
Wilson, P. A., and Melton, D. A. (1994) Curr Biol 4, 676-86.
Zimmerman, L. B., et al. (1996) Cell 86, 599-606.
The invention provides methods and compositions relating to DAN (Differential-screening-selected gene Aberrative in Neuroblastoma) and gremlin proteins and related nucleic acids. Included are natural DAN and gremlin homologs from different species and DAN and gremlin proteins comprising a DAN or gremlin domain and having DAN or gremlin-specific activity, particularly the ability to antagonize a bone morphogenic protein such as BMP2 or BMP4. The proteins may be produced recombinantly from transformed host cells with the subject nucleic acids. The invention provides isolated hybridization probes and primers capable of specifically hybridizing with the disclosed genes, specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for gremlin transcripts), therapy (e.g. gene therapy to modulate gremlin gene expression) and in the biopharmaceutical industry (e.g. reagents for screening chemical libraries for lead pharmacological agents).
Preferred applications of the subject DAN and gremlin proteins include modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DAN or gremlin protein under conditions whereby the added protein specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell. Also preferred are methods for screening for biologically active agents, which methods involve incubating a DAN or gremlin protein in the presence of an extracellular DAN or gremlin protein-specific binding target and a candidate agent, under conditions whereby, but for the presence of the agent, the protein specifically binds the binding target at a reference affinity; detecting the binding affinity of the protein to the binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the protein to the binding target.